Compositions of leukaemia inhibitory factor

ABSTRACT

The present invention relates generally to compositions and more particularly to compositions comprising leukaemia inhibitory factor (hereinafter referred to as “LIF”) or derivative or homologues thereof. The compositions of the present invention are particularly useful as compositions which exhibit enhanced stability and/or which exhibit reduced aggregation and/or reduced deamidation of LIF, its derivatives or other active ingredients.

FIELD OF THE INVENTION

The present invention relates generally to compositions and moreparticularly to compositions comprising leukaemia inhibitory factor(hereinafter referred to as “LIF”) or derivative or homologues thereof.The compositions of the present invention are particularly useful ascompositions which exhibit enhanced stability and/or which exhibitreduced aggregation and/or reduced deamidation of LIF, its derivativesor other active ingredients.

BACKGROUND OF THE INVENTION

LIF is a polyfunctional glycoprotein with diverse actions on a broadrange of tissue and cell types, including induction of differentiationin a number of myeloid leukaemic cell lines, suppression ofdifferentiation in normal embryonic stem cells, stimulation ofproliferation of osteoblasts and DA-1 haemopoietic cells andpotentiation of the of the proliferative action of interleukin-3 (IL-3)on megakaryocyte precursors. Functionally, LIF is able to switchautonomic nerve signalling from adrenergic to cholinergic mode,stimulate calcium release from bones, stimulate the production of acutephase proteins by hepatocytes and induce loss of fat deposits byinhibiting lipoprotein lipase-mediated lipid transport into adipocytes.

With a potentially broad range of clinical applications, it isimperative that compositions containing LIF are presented in a stableform and remain so during an extended period which may include shipment,handling and storage. Thus, a stable composition is one which retainsits physical, chemical, therapeutic and toxicological profile over thisperiod.

Deamidation is the most significant chemical degradation of LIF overtime. It is clearly desirable that this process is minimized. Adsorptionof LIF onto surfaces of containers, vials, syringes and infusion tubingis also a potential problem and must be minimized to ensure accuratedose and concentration. Physical degradation, such as aggregation orflocculation, may occur due to denaturation caused by elevatedtemperatures and/or agitation and excessive handling of the composition.Such degradation is clearly undesirable in terms of appearance and moreimportantly, consistent and effective administration of LIF in clinicalapplications. Storage at temperatures below room temperature typicallyretards chemical degradation, with storage in the frozen state beinggenerally the most effective. Whilst this may minimize chemicaldegradation, the process of thawing the composition may then result inaggregation.

Thus, there exists a need for a stable composition and, in particular, astable pharmaceutical composition of LIF and/or its derivatives orhomologues wherein chemical and physical degradation is minimised.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

One aspect of the present invention contemplates a compositioncomprising leukaemia inhibitory factor (LIF) or a derivative orhomologue thereof and a stabilizing agent facilitating chemical and/orphysical stability of LIF in the composition, additives for maintainingpH and isotonicity and one or more pharmaceutically acceptable carriersand/or diluents.

Another aspect of the invention provides a composition with improvedchemical and physical stability comprising LIF or a derivative orhomologue thereof, a stabilizing agent, additives for maintaining pH andisotonicity and one or more pharmaceutically acceptable carriers ordiluents under conditions in which aggregation of LIF is reduced.

Yet another aspect of the invention provides a composition with improvedchemical and physical stability comprising LIF or a derivative orhomologue thereof, a stabilizing agent, additives for maintaining pH andisotonicity and one or more pharmaceutically acceptable carriers ordiluents under conditions in which deamidation of LIF is reduced.

Still another aspect the present invention is directed to a stablecomposition comprising LIF or a derivative or homologue thereof,together with one or more pharmaceutically acceptable carriers ordiluents, wherein the composition has a pH of between about 3.5 andabout 6.5.

A further aspect the present invention provides a stable compositioncomprising LIF or a derivative or homologue thereof, together with oneor more pharmaceutically acceptable carriers or diluents, wherein thecomposition has a pH of between about 3.5 and about 6.5 under conditionsin which aggregation of LIF is reduced.

Another aspect the present invention contemplates a stable compositioncomprising LIF or a derivative or homologue thereof, together with oneor more pharmaceutically acceptable carriers or diluents, wherein thecomposition has a pH of between about 3.5 and about 6.5 under conditionsin which deamidation of LIF is reduced.

Yet another aspect of the present invention contemplates a method forpreparing a composition comprising Leukaemia Inhibition Factor (LIF) ora derivative or homologue thereof and which exhibits reduced deamidationand/or aggregation of LIF or a derivative or homologue over time saidmethod comprising admixing LIF or its derivative or homologue with astabilizing agent.

Still another aspect of the present invention is directed to the use ofa stabilizing agent in the manufacture of a composition exhibitingimproved chemical and/or physical stability of Leukaemic InhibitoryFactor (LIF) or a derivative or homologue thereof.

Preferred compositions in accordance with the present invention arereferred to as “pharmaceutical compositions” where LIF or itsderivatives or homologues is/are present in a pharmaceuticallyacceptable composition.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 to 3, respectively, are a diagrammatic representations ofReversed Phase, Ion Exchange and Size Exclusion chromatograms for a 1.0mg/ml standard solution of LIF prepared as described in Example 1 bydiluting “stock” solution with 2 mM phosphate buffer, pH 6.42,containing 0.01% polysorbate.

FIGS. 4 a–4 e are graphical representations showing LIF concentrationfor samples at each pH after freeze/thaw cycling.

FIG. 5 is a graphical representation of the average concentration over 5freeze/thaw cycles for each pH value.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides compositions comprising LIF or itsderivatives or homologues. The present invention particularly providesLIF or related molecules in a stable form.

Unless otherwise specified, the term “LIF” or “Leukaemia InhibitoryFactor” refers herein to synthetic, recombinant or purified naturallyoccurring LIF from animal or avian species. Preferred animal species aremammals such as humans, primates and livestock animals as well as any orall derivatives or homologues of LIF (e.g. sheep, pigs, cows, goats,donkeys and horses), laboratory animals (e.g. murine species, guineapigs, rabbits and hamsters), companion animals (e.g. dogs and cats) orcaptive wild animals (e.g. kangaroos, foxes, and deer). Preferred avianspecies include but are not limited to caged birds, chickens, ducks,geese and game birds. As referred to here, LIF or Leukaemia InhibitoryFactor includes reference to derivatives, homologues and analogues ofLIF. Derivatives, homologues, mimetics and analogues include parts,fragments or portions of LIF which are functionally active or whichotherwise have a useful biological activity (eg. as an antagonist,antigen to induce antibody formation, as a diagnostic agent or as atherapeutic molecule). Such derivatives or parts thereof include any oneor more contiguous series of amino acids contained within any one of theabove LIF molecules and includes single or multiple amino acidssubstitutions, deletions and/or additions to or in the natural,synthetic or recombinant LIF molecule as well as hyperglycosolated anddeglycosolated forms. Conditions for preparing recombinant LIF aredisclosed in International Patent Application Nos PCT/AU88/00093 andPCT/AU90/00001 although these conditions may vary depending on the hostcell used. Any such variations and/or modifications are within the scopeof the subject invention. The host cells may be eukaryotic (eg. yeast,mammalian, insect, plant etc) or prokaryotic (eg. Escherichia coli,Bacillus sp, Pseudomonas sp etc) cells.

Accordingly, one aspect of the present invention contemplates acomposition comprising leukaemia inhibitory factor (LIF) or a derivativeor homologue thereof and a stabilizing agent facilitating chemicaland/or physical stability of LIF in the composition, additives formaintaining pH and isotonicity and one or more pharmaceuticallyacceptable carriers and/or diluents.

Another aspect of the present invention provides a composition withimproved chemical and physical stability comprising LIF or a derivativeor homologue thereof, a stabilizing agent, additives for maintaining pHand isotonicity and one or more pharmaceutically acceptable carriers ordiluents under conditions in which aggregation of LIF is reduced.

Still yet another aspect of the present invention provides a compositionwith improved chemical and physical stability comprising LIF or aderivative or homologue thereof, a stabilizing agent, additives formaintaining pH and isotonicity and one or more pharmaceuticallyacceptable carriers or diluents under conditions in which deamidation ofLIF is reduced.

Analogues and mimetics include molecules which contain non-naturallyoccurring amino acids or which do not contain amino acids butnevertheless behave functionally the same as or similar to LIF. Naturalproduct screening is one useful strategy for identifying analogues andmimetics. Analogues of LIF contemplated herein also includemodifications to side chains, incorporation of unnatural amino acidsand/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on theprotein molecule or their analogues.

Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonicacid (TNBS); acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitisation, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carboethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringprotein synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids. A list of unnaturalamino acid contemplated herein is shown in Table 1.

TABLE 1 Non-conventional Non-conventional amino acid Code amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmglncarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-axnino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleuα-napthylalanine Anap D-α-methyllysine Dmlys N-benzylglycine NpheD-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine NglnD-α-methylornithine Dmorn N-(carbamylmethyl)glycine NasnD-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine NgluD-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp D-α-methylserineDmser N-cyclobutylglycine Ncbut D-α-methylthreonine DmthrN-cycloheptylglycine Nchep D-α-methyltryptophan DmtrpN-cyclohexylglycine Nchex D-α-methyltyrosine Dmty N-cyclodecylglycineNcdec D-α-methylvaline Dmval N-cylcododecylglycine NcdodD-N-methylalanine Dnmala N-cyclooctylglycine Ncoct D-N-methylarginineDnmarg N-cyclopropylglycine Ncpro D-N-methylasparagine DnmasnN-cycloundecylglycine Ncund D-N-methylaspartate DnmaspN-(2,2-diphenylethyl)glycine Nbhm D-N-methylcysteine DnmcysN-(3,3-diphenylpropyl)glycine Nbhe D-N-methylglutamine DnmglnN-(3-guanidinopropyl)glycine Narg D-N-methylglutamate DnmgluN-(1-hydroxyethyl)glycine Nthr D-N-methylhistidine DnmhisN-(hydroxyethyl))glycine Nser D-N-methylisoleucine DnmileN-(imidazolylethyl))glycine Nhis D-N-methylleucine DnmleuN-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine DnmlysN-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine NmchexaD-N-methylmethionine Dnmmet D-N-methylornithine DnmornN-methylcyclopentylalanine Nmcpen N-methylglycine NalaD-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate NmaibD-N-methylproline Dnmpro N-(1-methylpropyl)glycine Nile D-N-methylserineDnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methylα-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycinecarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbcethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilise 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH₂)_(n) groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety such as maleimido or dithiomoiety(SH) or carbodiimide(COOH). In addition, peptides can beconformationally constrained by, for example, incorporation of C_(α) andN_(α)-methylamino acids, introduction of double bonds between C_(α) andC_(β) atoms of amino acids and the formation of cyclic peptides oranalogues by introducing covalent bonds such as forming an amide bondbetween the N and C termini, between two side chains or between a sidechain and the N or C terminus.

All these types of modifications may be important to further stabiliseLIF in the composition of the present invention.

The compositions of the present invention achieve their stabilitythrough judicious choice of pH conditions within the range of from about3.5 to about 6.5 inclusive and optionally the presence of one or moresuitable stabilizing agents and/or additives. Preferably, the pH rangeis between from about 4.0–6.0 inclusive, more preferably between fromabout 4.5 to about 5.5 inclusive. Most preferably, the pH of thecomposition is about 5.0.

Accordingly, another aspect of the present invention provides acomposition comprising Leukaemia Inhibitory Factor (LIF) and one or morepharmaceutically acceptable carriers and/or diluents and wherein thecomposition has a pH of between about 3.5 and 6.5.

Suitable stabilizing agents are known to those skilled in the art andinclude agents which increase or maintain the conformational stabilityof LIF and surfactants. It is understood that one agent may possess morethan one stabilizing property and more than one agent may be employed toachieve a stabilizing effect.

Suitable agents are those which maintain approximately the same osmoticpressure as that of cellular fluids, and are known to those skilled inthe art. These may include, but are not limited to, polyhydric alcoholssuch as sorbitol, pharmaceutically acceptable salts such as NaCl, bufferspecies, sugars and pharmaceutically acceptable polymeric compounds.Suitable surfactants may be anionic, cationic, amphoteric or non-ionic.Preferred surfactants include fatty alcohols such as lauryl, cetyl andstearyl alcohols, glyceryl esters such as the mono-, di- andtriglycerides, fatty acid esters of fatty alcohols and esters of otheralcohols such as propylene glycol, polyethylene glycol, sorbitol,sucrose and cholesterol. Other suitable agents include the polysorbatessuch as polysorbates 20, 40, 60 and 80 and sorbitan ester,polyoxyethylene derivatives and pharmaceutically acceptablepolyoxyethylene-polyoxypropylene copolymers. Suitable agents whichmaintain or increase the conformational stability of LIF are also knownto the person skilled in the art and include sugars and polyhydricalcohols.

Suitable buffers for attaining the desired pH of the composition will beknown to those skilled in the art and include phosphate, citrate andacetate buffers. Preferred buffers are citrate and acetate.

Yet another aspect of the present invention contemplates a method ofpreparing a composition comprising Leukaemia Inhibitory Factor or aderivative or homologue thereof and which exhibits reduced deamidationand/or aggregation of LIF or a derivative or homologue over time saidmethod comprising admixing LIF or its derivative or homologue with astabilizing agent.

The compositions of the present invention may be suitable foradministration in a variety of forms such as, but not limited to,parenteral (e.g. intravenous, intraperitoneal, intramuscular,intradermal), subcutaneous, nasal, rectal, vaginal, topical, buccal andsublingual.

The carrier and other ingredients of the composition must bepharmaceutically “acceptable” in the sense of being compatible with theother ingredients of the composition and not injurious to the subject.The compositions may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Suchmethods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then if necessaryshaping the product.

Compositions of the present invention suitable for oral administrationmay be presented as a solution an aqueous or non-aqueous liquid; or asan oil-in-water liquid emulsion or a water-in-oil liquid emulsion. Theactive ingredient may also be presented as a bolus, electuary or paste.

Compositions suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured base, usuallysucrose and acacia or tragacanth gum; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia gum; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Compositions for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter.

Compositions suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Compositions suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containanti-oxidants, buffers, preservatives and solutes which render thecomposition isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The compositions may be presented inunit-dose or multi-dose sealed containers, for example, ampoules andvials, and may be stored in a freeze-dried (lyophilised) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

Preferred unit dosage compositions are those containing a daily dose orunit, daily sub-dose, as herein above described, or an appropriatefraction thereof, of the active ingredient.

It is also understood that the compositions of the present invention mayalso comprise one or more active agents or ingredients such a cytokinese.g. interleukins, CD antigens, colony stimulating factors, interferonsand tissue necrosis factor.

It should be understood that in addition to the active ingredientsparticularly mentioned above, the compositions of this invention mayinclude other agents conventional in the art having regard to the typeof composition in question, for example, those suitable for oraladministration may include such further agents as binders, sweeteners,thickeners, flavouring agents disintegrating agents, coating agents,preservatives, lubricants and/or time delay agents. Suitable sweetenersinclude sucrose, lactose, glucose, aspartame or saccharine. Suitabledisintegrating agents include corn starch, methylcellulose,polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar.Suitable flavouring agents include peppermint oil, oil of wintergreen,cherry, orange or raspberry flavouring. Suitable coating agents includepolymers or copolymers of acrylic acid and/or methacrylic acid and/ortheir esters, waxes, fatty alcohols, zein, shellac or gluten. Suitablepreservatives include sodium benzoate, vitamin E, alpha-tocopherol,ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite.Suitable lubricants include magnesium stearate, stearic acid, sodiumoleate, sodium chloride or talc. Suitable time delay agents includeglyceryl monostearate or glyceryl distearate.

A number of formulations of LIF were investigated in order to establishoptimum conditions under which chemical and physical degradation isreduced compared to the currently employed formulation of 3.67 mg/ml in2 mM phosphate buffer, pH 6.4–6.8.

Ion Exchange (IE), Reversed Phase (RP) and Size Exclusion (SEC)chromatography were used to detect changes in chemical and physicaldegradation.

Freeze/thaw studies revealed high solubility of LIF, i.e. noaggregation, in formulations in the pH range of 4.0–6.0 examined, thehighest being in the pH range of 4.5 to 5.5, with optimized stability atpH 5.0.

Studies of the various solutions over varying periods of storage time (0to 8 weeks) and at a range of storage temperatures (−80 to 25° C.)revealed optimum stability of the solution was achieved in a preferredpH range of 4.5 to 5.5.

The inventors examined a number of pH levels and additives. Samples atpH 4.0, 4.5, 5.0, 5.5 and 6.0 were prepared in Examples 1 and 2, asdescribed hereinafter, and additional additives, Sorbitol, anisotonicity agent, and Polysorbate 80 (also referred to as Tween-80), asa non-ionic surfactant to reduce non-specific adsorption onto surfaces,including glass, were also included. NaCl was also examined as anisotonicity agent.

LIF is present in the compositions of the invention in effectiveamounts. Effective amounts include from 0.1 mg/ml to 100 mg/ml.Preferred effective amounts are from 10 mg/ml to 10 mg/ml. Particularlypreferred amounts range from 400 mg/ml to 1000 mg/ml.

Suitable amounts of surfactant and isotonic agents may range from 0.001to 30%. Preferably from 0.01 to 10%, even more preferably from 0.01 to5.0%.

Particularly preferred compositions are those comprising LIF, sorbitol,polysorbate and a citrate or acetate buffer in the preferred rangesdescribed above.

The present invention further provides for the use of a stabilizingagent in the manufacture of a composition exhibiting improved chemicaland/or physical stability of Leukaemia Inhibitory Factor (LIF) or aderivative or homologue thereof.

The invention will now be described with reference to the followingnon-limiting Examples.

EXAMPLE 1

I. Preliminary Formulation Screening

On the basis of preliminary stability data, it was anticipated thatdeamidation of LIF would represent the principal pathway for degradationof solutions at neutral to slightly alkaline pH. Solution pH was,therefore, considered to be important and was a primary variableevaluated in these stability studies. Screening studies evaluating LIFstability during freeze/thaw cycling, following filtration, upon contactwith vials and syringes and following temperature controlled storagewere conducted in the pH range of 4 to 6 using acetate and citratebuffers at low concentrations (10 mM for each). Osmolality wascontrolled by the addition of sorbitol at a concentration of 5% w/v. Tominimise the potential for LIF adsorption to vials, filters, andsyringes, 0.01% w/v Polysorbate 80 was added to all preliminaryformulations evaluated in this series of studies.

II. Analytical Methods

Three analytical methods were used to assess LIF stability upon storage.A reversed phase assay, using a standard wide pore C8 reversed phasecolumn, was utilised for the purpose of total LIF concentrationdetermination. The reversed phase assay was not stability indicating andtherefore was not suitable for the determination of degradationproducts. A cation ion exchange assay was used to assess degradationproducts resulting from a change in the charge characteristics of theparent compound as deamidation had previously been determined to be theprincipal pathway for LIF degradation. A size exclusion assay was alsoused to detect size related changes (either cleavage, crosslinking, oraggregation) upon storage.

A. Reversed Phase (RP) Assay

Reversed phase chromatography was conducted using a wide pore C8reversed phase column, and a trifluoroacetic acid/acetonitrile mobilephase with gradient elution. Detection was conducted at 210 nm.

B. Ion-Exchange (IEC) Assay

Ion exchange chromatography was conducted using a cation exchangecolumn, pH 7 phosphate buffer and a salt gradient. Detection wasconducted at 280 nm.

C. Size Exclusion (SEC) Assay

Size exclusion chromatography was conducted using a dextrose based sizeexclusion column with a molecular weight range of 10 to 300 Daltons. Themobile phase was a pH 7.2 phosphate buffer and detection was conductedat 210 nm.

III. Method Validation

A. Reversed Phase (RP) Assay

Using the defined RP conditions, LIF eluted as a sharp, symmetrical peakwith a retention time of approximately 37 min as shown in FIG. 1. The RPassay was used for quantitation of total LIF only as the method was notselective for LIF in the presence of degradation (deamidation ordimeric) products.

Calibration curves for total peak area versus LIF concentration wereprepared with each set of analyses in the concentration range of 0.2 and1.0 mg/ml LIF.

Precision was determined from the coefficient of variation (CV, %) forthe total peak area obtained for replicate injections of standardsolutions prepared at 0.4 and 1.0 mg/ml. Accuracy was determined bycomparison of the total peak area for these standard solutions to aseparately prepared calibration curve and was expressed as thepercentage deviation from the nominal concentration. Results foraccuracy and precision with the RP assay are shown in Table 1. A summaryof the RP calibration curves is shown in Table 2.

B. Ion-Exchange (IEC) Assay

Using the defined IEC conditions, LIF eluted as a slightly tailing peakwith a retention time of approximately 13 min as shown in FIG. 2.Separation of the main LIF peak from degradation (deamidation) productsformed following storage was observed during the course of the studies.The actual identity of the degradation products (i.e. site ofdeamidation) was not determined in these studies.

Calibration curves for total peak area (main peak plus degradationproducts) versus LIF concentration were prepared with each set ofanalyses in the concentration range of 0.2 and 1.0 mg/ml LIF.Calibration curves were linear in this range when 100 μl was injectedonto the column.

Precision was determined from the coefficient of variation (CV, %) forthe total peak area obtained for replicate injections of standardsolutions prepared at 0.4 and 1.0 mg/ml. Accuracy was determined bycomparison of the total peak area for these standard solutions to aseparately prepared calibration curve and was expressed as thepercentage deviation from the nominal concentration. Results forprecision and accuracy for the IEC assay are shown in Table 3. A summaryof the IEC calibration curves over the course of the study is shown inTable 4.

C. Size Exclusion (SEC) Assay

Using the defined SEC conditions, LIF eluted as a sharp, symmetricalpeak with a retention time of approximately 26 min as shown in FIG. 3.The method separated monomeric LIF from dimeric LIF which eluted atapproximately 21 min, but was not selective for other degradation(deamidation) products which eluted as monomeric LIF.

Calibration curves for total peak area (main peak plus degradationproducts) versus LIF concentration were prepared with each set ofanalyses in the concentration range of 0.2 and 1.0 mg/mil LIF.

Precision was determined from the coefficient of variation (CV, %) forthe total peak area obtained for replicate injections of standardsolutions prepared at 0.4 and 1.0 mg/ml. Accuracy was determined bycomparison of the total peak area for these standard solutions to aseparately prepared calibration curve and was expressed as thepercentage deviation from the nominal concentration. Results forprecision and accuracy for the SEC assay are shown in Table 5. A summaryof the SEC calibration curves is shown in Table 6.

IV. Buffer Composition

All LIF samples were prepared by dilution of stock LIF solutioncontaining 3.67 mg/ml LIF in 2 mM phosphate buffer, pH 6.42 to give thedesired final LIF concentration (either 0.4 or 1.0 mg/ml) andcomposition of buffer components. In these studies, the finalcomposition of each solution contained 10 mM buffer (either acetate orcitrate), 5% w/v sorbitol and 0.01% w/v Polysorbate 80. Samples differedin the final concentration of phosphate buffer (present from theoriginal stock LIF solution) depending on the dilution factor. The 0.4mg/ml LIF solutions contained 0.22 mM residual phosphate while the 1.0mg/ml LIF solutions contained 0.54 μM residual phosphate. Thecomposition of each buffer was as follows:

A. Acetate Buffer for 0.4 mg/ml LIF Formulations

-   -   Solution A:        -   11.22 mM sodium acetate trihydrate (Merck #1.06267)        -   5.61% w/v sorbitol (Sigma Chemicals #S1876)        -   0.0112% w/v Polysorbate 80 (Sigma Chemicals #P1754)    -   Solution B:        -   11.22 mM glacial acetic acid (Sigma Chemicals #A6283)        -   5.61% w/v sorbitol (Sigma Chemicals #S1876)        -   0.0112% w/v Polysorbate 80 (Sigma Chemicals #P1754)

Solutions A and B were mixed to give a final pH of 4.0 or 4.5.Formulations were prepared by combining 0.109 parts stock LIF solutionand 0.891 parts buffer to give a final LIF concentration of 0.4 mg/ml, afinal buffer concentration of 10 mM, a final sorbitol concentration of5% w/v and a final Polysorbate 80 concentration of 0.01% w/v.

B. Acetate Buffer for 1.0 mg/ml LIF Formulations

-   -   Solution A:        -   13.75 mM sodium acetate trihydrate (Merck #1.06267)        -   6.88% w/v sorbitol (Sigma Chemicals #S1876)        -   0.0138% w/v Polysorbate 80 (Sigma Chemicals #P1754)    -   Solution B:        -   13.75 mM glacial acetic acid (Sigma Chemicals #A6283)        -   6.88% w/v sorbitol (Sigma Chemicals #S1876)        -   0.0138% w/v Polysorbate 80 (Sigma Chemicals #P1754)

Solutions A and B were mixed to give a final pH of 4.0 or 4.5.Formulations were prepared by combining 0.272 parts stock LIF solutionand 0.728 parts buffer to give a final LIF concentration of 1.0 mg/ml, afinal buffer concentration of 10 mM, a final sorbitol concentration of5% w/v and a final Polysorbate 80 concentration of 0.01% w/v.

C. Citrate Buffer for 0.4 mg/ml LIF Formulations

-   -   Solution A:        -   11.22 mM sodium citrate dihydrate (Merck #1.06448)        -   5.61% w/v sorbitol (Sigma Chemicals #S1876)        -   0.0112% Polysorbate 80 (Sigma Chemicals #P1754)    -   Solution B:        -   11.22 mM citric acid monohydrate (Merck #1.00244)        -   5.61% w/v sorbitol (Sigma Chemicals #S1876)        -   0.0112% Polysorbate 80 (Sigma Chemicals #P1754)

Solutions A and B were mixed to give a final pH of 5.0, 5.5, or 6.0.Formulations were prepared by combining 0.109 parts stock LIF solutionand 0.891 parts buffer to give a final LIF concentration of 0.4 mg/ml, afinal buffer concentration of 10 mM, a final sorbitol concentration of5% w/v and a final Polysorbate 80 concentration of 0.01% w/v.

D. Citrate Buffer for 1.0 mg/ml LIF Formulations

-   -   Solution A:        -   13.75 mM sodium citrate (Merck #1.06448)        -   6.88% w/v sorbitol (Sigma Chemicals #S1876)        -   0.0138% w/v Polysorbate 80 (Sigma Chemicals #P1754)    -   Solution B:        -   13.75 mM citric acid (Merck #1.00244)        -   6.88% w/v sorbitol (Sigma Chemicals S1876)        -   0.0138% w/v Polysorbate 80 (Sigma Chemicals P1754)

Solutions A and B were mixed to give a final pH of 5.0, 5.5, or 6.0.Formulations were prepared by combining 0.272 parts stock LIF solutionand 0.728 parts buffer to give a final LIF concentration of 1.0 mg/ml, afinal buffer concentration of 10 mM, a final sorbitol concentration of5% w/v and a final Polysorbate 80 concentration of 0.01% w/v.

Table 7 displays pH and osmolality (obtained using a Fiske One-TenOsmometer) values for 0.4 and 1.0 mg/mil LIF samples prepared using theabove buffer systems.

V. Freeze/Thaw Cycling

A. Sample Preparation and Methods

LIF samples were prepared by dilution of stock LIF (3.67 mg/ml in 2 mMphosphate buffer, pH 6.8) with acetate or citrate buffer containingsorbitol and polysorbate 80 to give a final buffer concentration of 10mM, a theoretical pH of 4.0, 4.5, 5.0, 5.5, or 6.0, a final sorbitolconcentration of 5% w/v, a final polysorbate 80 concentration of 0.01%w/v and a final LIF concentration of 1 mg/ml (see Section IV). The finalpH of each sample was essentially the same as predicted by theory.Solutions (3 ml) were filtered through 0.22 μm sterile filters (MillexGV) with the first 0.5 ml aliquot from the filter being retained as aseparate sample for the preliminary determination of filter adsorption.Subsequent 0.5 ml aliquots were filtered into sterile 2 ml glass vialsand capped with sterile rubber/teflon lined caps and crimped. One vialfor each formulation was analysed on the day of preparation and allother vials were stored at −80° C. On each of 5 days, all vials werethawed and one vial of each formulation was centrifuged and an aliquottaken for dilution (in this study, all samples were analysed at a LIFconcentration of 0.1 mg/ml) and analysis by RP, IEC, and SEC.

A 0.1 mg/ml standard solution was prepared by diluting the LIF stocksolution with 2 mM phosphate buffer, pH 6.42 containing 0.01%polysorbate 80. This standard solution was stored at 4° C. for a totalof 6 days and analysed along with each sample set.

B. Results

FIGS. 4 a–4 e represent the individual results for samples at each pHwith concentration being expressed as a percentage of the initialconcentration measured by each of the three methods.

While there was some variability in the individual results (most likelydue to the dilution step prior to analysis), there were no trends whichwould indicate loss of LIF upon freeze/thaw cycling.

FIG. 5 illustrates the average concentration (as a percentage of theinitial concentration) over 5 freeze/thaw cycles for each of thedifferent pH values.

VI. Long Term Stability at −80° C., −20° C., 8° C. and 25° C.

A. Preparation of Samples for Storage at −80° C. and −20° C.

Five LIF formulations were prepared by dilution of stock LIF (3.67 mg/mlin 2 mM phosphate buffer, pH 6.42) with acetate or citrate buffercontaining sorbitol and polysorbate 80 to give a final LIF concentrationof 0.4 mg/ml or 1.0 mg/ml, a final buffer concentration of 10 mM, afinal sorbitol concentration of 5% w/v and a final polysorbate 80concentration of 0.01% w/v (see Section V). The theoretical pH valueswere pH 4.0 (acetate buffer), 4.5 (acetate buffer), and 5.0 (citratebuffer). The final pH of each sample was essentially the same aspredicted by theory.

Under aseptic conditions in a laminar flow cabinet, the formulationswere sterile filtered using 0.22 μm Millex GV (Millipore) filters. Thefirst 1.0 ml of each filtrate was set aside and the vial markedaccordingly (previous studies identified that approximately 1 ml wasrequired to saturate the filter binding sites using Millex GV filterunits). The remaining volume was filtered into a sterile 50 mlpolypropylene tube. Aliquots of each formulation (1.15 ml/vial) weretransferred using a multiple dispensing Eppendorf pipette with steriletips into heat sterilised 2 ml glass vials and capped with sterileteflon lined rubber caps which were then crimped. Vials were labelledand duplicate vials of each formulation were retained for the initialanalysis. The remaining vials were stored at either −80° C. or −20° C.

B. Preparation of Samples for Storage at 8° C. and 25° C.

Five LIF formulations were prepared by a dilution of stock LIF (3.67mg/ml in 2 mM phosphate buffer, pH 6.42) with acetate or citrate buffercontaining sorbitol and polysorbate 80 to give a final LIF concentrationof 0.4 mg/ml or 1.0 mg/ml, a final buffer concentration of 10 mM, afinal sorbitol concentration of 5% w/v and a final polysorbate 80concentration of 0.01% w/v. The theoretical pH values were pH 4.0(acetate buffer), 4.5 (acetate buffer), and 5.0 (citrate buffer). Thefinal pH of each sample was essentially the same as predicted by theory.

Formulations were filtered and filled into vials as described for the−80° C. and −20° C. samples. Samples were stored in temperaturecontrolled incubators at either 8° C. or 25° C. Incubators were checkeddaily to ensure the correct temperature was maintained.

C. Sample Analysis

All LIF samples were analysed undiluted according to the methodsdescribed in Section m. LIF standards at concentrations of 0.2, 0.4, 0.7and 1.0 mg/ml were prepared from stock LIF (3.67 mg/ml in 2 mM phosphatebuffer) by diluting with 2 mM phosphate buffer, pH 6.42 containing 0.01%w/v polysorbate 80. These standards were prepared fresh at the beginningof each set of analyses and were analysed along with the samples at thestart and end of each analytical run.

At each time point, 2 vials were withdrawn from the freezers orincubators and approximately 200 μl was removed from each using asterile 1 ml syringe and a sterile needle. These aliquots were placedinto polypropylene autosampler vials and sealed with caps containingself-sealing septa to allow repeat injections from the same vial withoutevaporation.

Autosampler vials were transferred to the autosampler where they weremaintained at 4° C. throughout the three analytical runs. The samesample and standard autosampler vials were used for each of the threeanalyses with the RP (10 μl injection volume) being conducted first,followed by the IEC (100 μl injection volume) and then the SEC (10 μlinjection volume). The complete RP run took approximately 32 hours, andthe IEC and SEC runs took approximately 25 hours each. It was assumedthat any further degradation over this storage time in the autosamplerwould be minimal (standard solutions at pH 6.42 stored under the sameconditions showed no change over the complete analytical period).Samples were analysed in the following order:

Blank × 2 0.2 mg/ml, 0.4 mg/ml, 0.7 mg/ml and 1.0 mg/ml Standards Blank0.4 mg/ml Acetate pH 4.0 × 2 Acetate pH 4.5 × 2 Citrate pH 5.0 × 2 1.0mg/ml Acetate pH 4.5 × 2 Citrate pH 5.0 × 2 Blank 0.2 mg/ml, 0.4 mg/ml,0.7 mg/ml and 1.0 mg/ml Standards

Selected samples were also analysed for particulates using a MalvernInstruments Zetasizer 3000 particle size instrument. Samples werewithdrawn from the storage vials using a syringe and placed in thesample cuvette. Samples were counted for 120 sec using a 200 μm pinhole(to obtain the maximum signal), 90° scattering angle, and scatteringsource at 633 nm using a 10 mW He—Ne ion laser.

D. Results

Data pertaining to solution pH, LIF concentration in mg/ml (determinedby comparison to LIF standard solutions), and the area % of the mainpeak relative to the total peak area for all LIF related peaks in thechromatogram analysed using the three chromatographic methods are shownin Tables 8 through 17. None of the samples showed significant shifts inpH over the storage period.

1. Ion Exchange

IEC chromatograms for samples stored in each of the different buffersystems at 8 and 25° C. showed two main products for samples prepared inpH 4.0 and 4.5 buffers (eluting at approximately 9 and 10 min) whereas asingle main product (eluting at approximately 10 min) was seen in the pH5.0 samples. At each pH, there was evidence of several minor degradationproducts in the ion exchange chromatograms, however, due to inadequateresolution between the different products, the exact number of productscould not be determined. Representative chromatograms for samples storedat −80 and −20° C. are not shown as they were similar to thechromatograms at the higher temperatures with degradation products beingpresent at significantly reduced levels.

The IEC results for samples stored at −80, −20, 8 and 25° C. illustratethe dependence of LIF stability on pH and temperature (Tables 8–17). Therelative stability under each storage condition was similar for the 0.4and 1.0 mg/ml formulations. The pH 4.0 samples displayed significantvariability between the different time points at 8 and 25° C.Re-analysis of selected samples gave similar results to the originalvalues. There was also evidence of degradation at pH 4.0 and 4.5following storage at −20° C. and −80° C. The stability was greatlyimproved at pH 5 in comparison to pH 4 and 4.5. At pH 5.0 after 55 daysstorage at 8° C., approximately 97% of the total peak area was presentas the main LIF peak. Following storage at 25° C. for 55 days, thisvalue was reduced to approximately 78%. Samples prepared at pH 5 andstored at −80 or −20° C. for up to 84 days showed no significantevidence of degradation.

2. Reversed Phase

Representative RP chromatograms are not included as all displayedessentially the same elution characteristics (see FIG. 1). In all cases,the chromatograms showed the presence of only one main peak eluting atapproximately 36 min.

The RP results for samples stored at −80, −20, 8 and 25° C., wherein themeasured concentration was plotted as a function of storage time,illustrated the absence of significant change in the measuredconcentration over the storage period for each of the buffer and storageconditions utilised.

3. Size Exclusion

SEC chromatograms for the samples as all displayed essentially the sameelution characteristics (see FIG. 3). In all cases, the chromatogramsshowed the presence of one main peak eluting at approximately 26 min anda minor peak eluting at approximately 21 min.

The SEC results for samples stored at −80, −20, 8 and 25° C. wherein themeasured concentration was plotted as a function of storage time,illustrated the absence of significant change in the measuredconcentration over the storage period for each of the buffer and storageconditions utilised. Using the SEC method, there was no evidence ofchain cleavage or crosslinking under the storage conditions studied.

4. Particle Size Analysis

Samples stored for 56 days at −80 and −20° C. and for 41 days at 8 and25° C. were analysed for particulates using a laser light scatteringinstrument. All of the samples analysed displayed a count rate of “0kCps” which effectively means that the samples contained no particulates(i.e. no signal was measurable).

VII. Summary

These studies demonstrated no notable loss of LIF following freeze thawcycling of 1.0 mg/ml LIF solution formulations prepared in acetate orcitrate buffers (pH 4 to 6) containing 5% w/v sorbitol and 0.01% w/vpolysorbate 80. There was no significant loss of LIF on 0.2 μm SartoriusMinisart filters when formulations were prepared at either 0.4 or 1.0mg/ml in pH 5.0 or 5.5 citrate buffers containing 5% w/v sorbitol and0.01% w/v polysorbate 80. For the pH 5.0 and 5.5 formulations, there wasalso no evidence of loss of LIF on the proposed vials, stoppers, orsyringes.

At −80° C., there was no significant change in LIF concentrationsmeasured by RP, IEC and SEC methods following storage for 84 days in thepH range of 4 to 5. At −20° C. over the same time period, there wasevidence of degradation for formulations prepared at pH 4 and analysedby EC, but the remaining formulations were stable under these storageconditions. Generally, 0.4 and 1.0 mg/ml LIF formulations displayedsimilar stability characteristics under each of the conditionsinvestigated. Formulations prepared at pH 5 were found to be stable forup to 8 weeks when stored at 8° C. with minimal loss of the parentcompound (˜1%) shown by IEC and no loss shown by RP or SEC.

TABLE 1 Precision and Accuracy for the RP Assay Nominal Conc. Total PeakMeasured Precision Accuracy (mg/ml) Area Conc. (mg/ml) (CV, %) (%deviation) 0.4 14.213 0.391 0.44 (n = 5) −2.16 0.4 14.356 0.395 −1.210.4 14.361 0.395 −1.17 0.4 14.322 0.394 −1.43 0.4 14.255 0.392 −1.88 1.038.002 1.029 0.39 (n = 5) 2.92 1.0 38.170 1.034 3.37 1.0 38.327 1.0383.79 1.0 38.344 1.038 3.84 1.0 38.077 1.031 3.12

TABLE 2 Summary of RP Calibration Curves Over the Course of the StudySlope Intercept 33.460 −1.755 32.900 −0.312 34.491 −1.040 32.648 −0.13732.865 1.006 32.865 0.566 33.705 1.092 34.617 0.535 35.920 0.113 35.666−0.014 37.294 −0.382 mean 34.221 −0.030 SD 1.529 CV, % 4.469

TABLE 3 Precision and Accuracy for the IEC Assay Nominal Conc. TotalPeak Measured Precision Accuracy (mg/ml) Area Conc. (mg/ml) (CV, %) (%deviation) 0.4 8.310 0.397 0.68 (n = 5) −0.86 0.4 8.260 0.398 −0.62 0.48.265 0.399 −0.30 0.4 8.232 0.396 −1.10 0.4 8.234 0.403 0.65 1.0 21.9291.007 0.41 (n = 5) 0.70 1.0 21.910 1.005 0.51 1.0 21.918 1.008 0.77 1.021.901 1.004 0.35 1.0 21.870 1.014 1.43

TABLE 4 Summary of IEC Calibration Curves Over the Course of the StudySlopes Intercept 2.953 −0.002 3.111 −0.038 3.104 −0.048 2.983 −0.0192.987 −0.020 3.005 −0.018 2.942 −0.012 3.064 −0.055 3.005 −0.018 3.034−0.036 3.137 −0.099 mean 3.030 −0.033 SD 0.066 — CV, % 2.180 —

TABLE 5 Precision and Accuracy for the SEC Assay Nominal Conc. TotalPeak Measured Precision Accuracy (mg/ml) Area Conc. (mg/ml) (CV, %) (%deviation) 0.4 8.310 0.396 0.39 (n = 5) −0.98 0.4 8.260 0.394 −1.48 0.48.265 0.394 −1.46 0.4 8.232 0.393 −1.84 0.4 8.234 0.393 −1.86 1.0 21.9291.002 0.11 (n = 5) 0.23 1.0 21.910 1.001 0.11 1.0 21.918 1.001 0.15 1.021.901 1.000 0.07 1.0 21.870 0.999 −0.05

TABLE 6 Summary of SEC Calibration Curves Over the Course of the StudySlope Intercept 21.332 0.202 21.278 0.166 22.351 0.230 21.672 0.05420.810 0.419 21.561 0.130 21.845 0.074 21.883 −0.090 21.963 0.158 21.794−0.003 22.558 −0.474 mean 21.732 0.079 SD 0.491 — CV, % 2.258 —

TABLE 7 pH and Osmolality of AM424 Formulations AM424 conc. osmolalitybuffer/theoretical pH (mg/ml) measured pH (mOsm/kg) Acetate/pH 4.0 0.43.95 297 Acetate/pH 4.5 0.4 4.48 297 Citrate/pH 5.0 0.4 4.94 303Acetate/pH 4.5 1.0 4.47 294 Citrate/pH 5.0 1.0 4.96 305

TABLE 8 Summary of 0.4 mg/ml, pH 4.0 AM424 Formulation StabilityFollowing Storage at 8° C. and 25° C. Nominal RP - IEC - SEC - AM424Storage Measured RP - Measured IEC - Measured SEC - measured Conc.Storage Time Conc. Main Peak Conc. Main Peak Conc. Main Peak pH buffer(mg/ml) Temp. (C.) (days) (mg/ml) (area %) (mg/ml) (area %) (mg/ml)(area %) 4.03 acetate 0.4 8 0 0.40, 0.39 100, 100 0.37, 0.37 98.9, 99.00.39, 0.40 98.8, 98.7 — 7 0.40, 0.40 100, 100 0.35, 0.34 91.7, 87.20.39, 0.40 97.6, 97.5 4.07 13 0.39, 0.39 100, 100 0.33, 0.37 90.8, 92.80.40, 0.40 98.9, 98.9 — 19 0.40, 0.40 100, 100 0.34, 0.33 89.7, 86.80.38, 0.38 98.8, 99.0 4.06 27 0.40, 0.40 100, 100 0.33, 0.33 84.6, 83.70.40, 0.40 98.9, 98.9 4.06 41 0.40, 0.40 100, 100 0.34, 0.35 86.9, 88.20.40, 0.41 98.9, 98.9 4.16 55 0.40, 0.41 100, 100 0.34, 0.33 89.2, 83.00.40, 0.40 99.0, 99.0 4.03 acetate 0.4 25 0 0.40, 0.39 100, 1000.37, 0.37 98.9, 99.0 0.39, 0.40 98.8, 98.7 — 7 0.39, 0.39 100, 1000.33, 0.36 85.1, 91.5 0.39, 0.40 97.3, 97.4 4.06 13 0.40, 0.39 100, 1000.28, 0.30 74.7, 80.7 0.39, 0.41 99.2, 99.1 — 19 0.40, 0.39 100, 1000.31, 0.32 78.3, 80.3 0.38, 0.38 99.0, 99.2 4.07 27 0.40, 0.40 100, 1000.29, 0.30 73.3, 74.5 0.40, 0.40 99.4, 99.2 4.09 41 0.40, 0.40 100, 1000.31, 0.31 76.1, 77.8 0.41, 0.41 99.2, 99.2 4.12 55 0.41, 0.40 100, 1000.25, 0.24 62.6, 59.8 0.40, 0.40 99.3, 99.7 Underlined values representrepeat analyses

TABLE 9 Summary of 0.4 mg/ml, pH 4.0 AM424 Formulation StabilityFollowing Storage at −80° C. and −20° C. Nominal RP - IEC - SEC - AM424Storage Measured RP - Measured IEC - Measured SEC - measured Conc.Storage Time Conc. Main Peak Conc. Main Peak Conc. Main Peak pH buffer(mg/ml) Temp. (C.) (days) (mg/ml) (area %) (mg/ml) (area %) (mg/ml)(area %) 3.95 acetate 0.4 −80 0 0.41, 0.40 100, 100 0.38, 0.38 98.4,98.6 0.40, 0.40 98.9, 98.5 3.98 28 0.41, 0.41 100, 100 0.38, 0.39 97.8,98.8 0.39, 0.40 98.2, 98.0 3.99 56 0.41, 0.41 100, 100 0.37, 0.38 96.6,98.9 0.39, 0.39 98.3, 98.3 4.05 84 0.43, 0.42 100, 100 0.40, 0.38 98.6,99.1 0.41, 0.41 99.3, 98.6 3.95 acetate 0.4 −20 0 0.41, 0.40 100, 1000.38, 0.38 98.4, 98.6 0.40, 0.40 98.9, 98.5 3.95 28 0.41, 0.42 100, 1000.38, 0.39 96.9, 97.8 0.40, 0.40 98.7, 98.5 4.04 56 0.40, 0.41 100, 1000.36, 0.36 94.2, 93.7 0.40, 0.40 99.0, 98.9 4.03 84 0.42, 0.43 100, 1000.38, 0.38 92.5, 93.1 0.42, 0.41 99.2, 98.9

TABLE 10 Summary of 0.4 mg/ml, pH 4.5 AM424 Formulation StabilityFollowing Storage at 8° C. and 25° C. Nominal RP - IEC - SEC - AM424Storage Measured RP - Measured IEC - Measured SEC - measured Conc.Storage Time Conc. Main Peak Conc. Main Peak Conc. Main Peak pH buffer(mg/ml) Temp. (C.) (days) (mg/ml) (area %) (mg/ml) (area %) (mg/ml)(area %) 4.52 acetate 0.4 8 0 0.39, 0.39 100, 100 0.36, 0.36 99.0, 98.90.39, 0.38 98.8, 98.8 — 7 0.38, 0.38 100, 100 0.37, 0.36 95.4, 95.60.38, 0.39 97.7, 97.8 4.53 13 0.38, 0.38 100, 100 0.38, 0.36 98.3, 95.60.39, 0.38 99.0, 99.0 — 19 0.38, 0.38 100, 100 0.37, 0.35 97.8, 93.30.38, 0.38 98.2, 98.2 4.53 27 0.38, 0.38 100, 100 0.35, 0.36 90.8, 94.10.39, 0.39 98.9, 98.8 4.51 41 0.39, 0.39 100, 100 0.37, 0.36 95.3, 94.20.39, 0.39 98.9, 98.8 4.59 55 0.40, —  100, —  0.35, 0.33 89.6, 85.90.39, 0.39 99.0, 98.9 4.52 acetate 0.4 25 0 0.39, 0.39 100, 1000.36, 0.36 99.0, 98.9 0.39, 0.39 98.8, 98.8 — 7 0.38, 0.38 100, 1000.36, 0.34 94.7, 88.8 0.39, 0.39 98.1, 98.2 4.52 13 0.39, 0.38 100, 1000.33, 0.35 86.8, 91.0 0.39, 0.38 99.0, 99.0 — 19 0.38, 0.38 100, 1000.31, 0.30 82.0, 80.0 0.38, 0.38 99.1, 99.0 4.52 27 0.38, 0.38 100, 1000.30, 0.29 75.8, 73.5 0.39, 0.39 99.1, 99.2 4.53 41 0.40, 0.40 100, 1000.28, 0.28 71.2, 71.1 0.39, 0.39 99.2, 99.3 4.55 55 0.39, 0.40 100, 1000.22, 0.24 53.4, 59.1 0.39, 0.39 99.3, 99.4 Underlined values representrepeat analyses

TABLE 11 Summary of 0.4 mg/ml, pH 1.5 AM424 Formulation StabilityFollowing Storage at −80° C. and −20° C. Nominal RP - IEC - SEC - AM424Storage Measured RP - Measured IEC - Measured SEC - measured Conc.Storage Time Conc. Main Peak Conc. Main Peak Conc. Main Peak pH buffer(mg/ml) Temp. (C.) (days) (mg/ml) (area %) (mg/ml) (area %) (mg/ml)(area %) 4.48 acetate 0.4 −80 0 0.40, 0.40 100, 100 0.39, 0.40 99.0,98.9 0.40, 0.40 98.9, 98.8 4.49 28 0.41, 0.40 100, 100 0.39, 0.38 98.7,98.7 0.40, 0.39 98.1, 98.0 4.49 56 0.40, 0.40 100, 100 0.38, 0.38 98.5,98.6 0.39, 0.39 98.4, 98.2 4.55 84 0.42, 0.42 100, 100 0.40, 0.40 98.6,98.4 0.42, 0.42 98.5, 98.5 4.48 acetate 0.4 −20 0 0.40, 0.40 100, 1000.39, 0.40 99.0, 98.9 0.40, 0.40 98.9, 98.8 4.47 28 0.41, 0.41 100, 1000.39, 0.38 98.8, 96.9 0.40, 0.40 98.4, 98.6 4.52 56 0.40, 0.40 100, 1000.39, 0.38 98.5, 97.3 0.40, 0.39 98.6, 98.7 4.53 84 0.42, 0.42 100, 1000.40, 0.40 96.4, 96.5 0.42, 0.42 99.0, 99.0

TABLE 12 Summary of 1.0 mg/ml, pH 4.5 AM424 Formulation StabilityFollowing Storage at 8° C. and 25° C. Nominal RP - IEC - SEC - AM424Storage Measured RP - Measured IEC - Measured SEC - measured Conc.Storage Time Conc. Main Peak Conc. Main Peak Conc. Main Peak pH buffer(mg/ml) Temp. (C.) (days) (mg/ml) (area %) (mg/ml) (area %) (mg/ml)(area %) 4.54 acetate 1.0 8 0 0.99, 0.99 100, 100 0.96, 0.96 98.5, 98.60.99, 0.99 98.6, 98.2 — 7 0.98, 0.98 100, 100 0.98, 0.99 96.9, 979 0.99, 0.99 98.6, 98.6 4.57 13 0.98, 0.99 100, 100 0.94, 0.96 96.2, 97.60.98, 0.98 98.8, 98.7 — 19 0.98, 1.00 100, 100 0.96, 0.94 97.5, 95.61.00, 0.99 98.6, 98.8 4.56 27 0.99, 1.00 100, 100 0.94, 0.88 97.0, 90.11.00, 1.00 98.6, 98.8 4.55 41 0.98, 0.99 100, 100 0.88, 0.90 90.3, 92.10.98, 0.98 98.9, 98.9 4.61 55 0.99, 1.00 100, 100 0.90, 0.85 91.2, 86.10.99, 0.99 98.9, 98.9 4.54 acetate 1.0 25 0 0.99, 0.99 100, 1000.96, 0.96 98.5, 98.6 0.99, 0.99 98.6, 98.2 — 7 0.99, 0.99 100, 1000.92, 0.94 91.4, 92.7 0.99, 0.99 98.9, 98.9 4.57 13 1.00, 0.99 100, 1000.82, 0.86 83.6, 86.6 0.98, 0.98 99.0, 99.0 — 19 1.00, 1.00 100, 1000.84, 0.81 83.7, 80.9 1.00, 1.00 98.9, 98.9 4.57 27 1.00, 1.00 100, 1000.78, 0.81 77.1, 79.3 1.00, 1.00 99.0, 99.0 4.59 41 0.99, 0.99 100, 1000.68, 0.65 66.4, 63.9 0.98, 0.98 98.9, 99.1 4.61 55 1.00, 0.99 100, 1000.59, 0.60 56.7, 58.9 0.99, 0.99 99.1, 99.1 Underlined values representrepeat analyses

TABLE 13 Summary of 1.0 mg/ml, pH 4.5 AM424 Formulation StabilityFollowing Storage at −80° C. and −20° C. Nominal RP - IEC - SEC - AM424Storage Measured RP - Measured IEC - Measured SEC - measured Conc.Storage Time Conc. Main Peak Conc. Main Peak Conc. Main Peak pH buffer(mg/ml) Temp. (C.) (days) (mg/ml) (area %) (mg/ml) (area %) (mg/ml)(area %) 4.47 acetate 1.0 −80 0 1.00, 1.00 100, 100 0.97, 0.98 98.9,98.7 0.99, 0.99 98.7, 98.6 4.47 28 1.00, 1.00 100, 100 0.97, 0.97 98.4,98.3 0.99, 0.99 98.4, 98.4 4.50 56 0.99, 0.98 100, 100 0.96, 0.95 98.5,98.3 0.97, 0.97 98.5, 98.6 4.53 84 1.00, 1.00 100, 100 0.96, 0.96 98.3,98.5 0.98, 0.98 98.6, 98.4 4.47 acetate 1.0 −20 0 1.00, 1.00 100, 1000.97, 0.98 98.9, 98.7 0.99, 0.99 98.7, 98.6 4.48 28 1.00, 0.99 100, 1000.98, 0.97 98.3, 97.4 1.00, 0.99 98.5, 98.6 4.50 56 0.98, 0.99 100, 1000.94, 0.96 97.4, 98.0 0.98, 0.98 98.6, 98.5 4.51 84 0.99, 0.99 100, 1000.95, 0.97 96.9, 98.4 0.99, 0.99 98.7, 98.5

TABLE 14 Summary of 0.4 mg/ml, pH 5.0 AM424 Formulation StabilityFollowing Storage at 8° C. and 25° C. Nominal RP - IEC - SEC - AM424Storage Measured RP - Measured IEC - Measured SEC - measured Conc.Storage Time Conc. Main Peak Conc. Main Peak Conc. Main Peak pH buffer(mg/ml) Temp. (C.) (days) (mg/ml) (area %) (mg/ml) (area %) (mg/ml)(area %) 5.02 citrate 0.4 8 0 0.38, 0.37 100, 100 0.36, 0.36 98.8, 98.70.38, 0.38 98.6, 98.4 — 7 0.37, 0.37 100, 100 0.38, 0.38 98.4, 98.40.37, 0.37 98.1, 98.1 5.03 13 0.37, 0.37 100, 100 0.38, 0.38 98.4, 98.40.38, 0.38 98.6, 98.7 — 19 0.37, 0.37 100, 100 0.37, 0.37 98.4, 98.50.37, 0.37 98.3, 98.0 5.06 27 0.38, 0.38 100, 100 0.38, 0.37 98.4, 98.50.38, 0.38 98.6, 98.6 5.04 41 0.39, 0.39 100, 100 0.38, 0.37 97.8, 97.90.38, 0.38 98.7, 98.7 5.07 55 0.39, 0.39 100, 100 0.37, 0.37 97.7, 97.50.39, 0.38 98.8, 98.7 5.02 citrate 0.4 25 0 0.38, 0.37 100, 1000.36, 0.36 98.8, 98.7 0.38, 0.38 98.6, 98.4 — 7 0.37, 0.37 100, 1000.37, 0.36 97.0, 97.0 0.38, 0.38 98.7, 98.5 5.05 13 0.38, 0.37 100, 1000.36, 0.37 95.4, 95.1 0.37, 0.37 98.7, 98.7 — 19 0.37, 0.37 100, 1000.35, 0.35 93.8, 93.9 0.38, 0.38 98.8, 98.8 5.05 27 0.38, 0.38 100, 1000.34, 0.34 92.0, 91.8 0.39, 0.39 99.3, 99.0 5.06 41 0.39, 0.39 100, 1000.33, 0.34 87.0, 87.4 0.38, 0.38 99.1, 99.1 5.03 55 0.39, 0.39 100, 1000.30, 0.30 77.8, 77.9 0.39, 0.39 99.0, 98.9 Underlined values representrepeat analyses

TABLE 15 Summary of 0.4 mg/ml, pH 5.0 AM424 Formulation StabilityFollowing Storage at −80° C. and −20° C. Nominal RP - IEC - SEC - AM424Storage Measured RP - Measured IEC - Measured SEC - measured Conc.Storage Time Conc. Main Peak Conc. Main Peak Conc. Main Peak pH buffer(mg/ml) Temp. (C.) (days) (mg/ml) (area %) (mg/ml) (area %) (mg/ml)(area %) 4.94 citrate 0.4 −80 0 0.41, 0.40 100, 100 0.40, 0.40 98.9,98.8 0.39, 0.39 98.8, 98.7 4.98 28 0.41, 0.41 100, 100 0.39, 0.39 98.5,98.5 0.39, 0.39 98.3, 98.3 4.98 56 0.40, 0.40 100, 100 0.38, 0.38 98.6,98.6 0.38, 0.38 98.4, 98.3 5.00 84 0.42, 0.42 100, 100 0.40, 0.40 98.7,98.4 0.41, 0.41 98.5, 98.5 4.94 citrate 0.4 −20 0 0.41, 0.41 100, 1000.40, 0.40 98.9, 98.8 0.39, 0.39 98.8, 98.7 4.95 28 0.41, 0.41 100, 1000.39, 0.39 98.5, 98.5 0.40, 0.40 98.5, 98.6 4.96 56 0.40, 0.40 100, 1000.38, 0.39 98.4, 98.6 0.39, 0.39 98.6, 98.6 4.97 84 0.42, 0.42 100, 1000.41, 0.41 98.6, 98.7 0.41, 0.41 99.0, 98.8

TABLE 16 Summary of 1.0 mg/ml, pH 5.0 AM424 Formulation StabilityFollowing Storage at 8° C. and 25° C. Nominal RP - IEC - SEC - AM424Storage Measured RP - Measured IEC - Measured SEC - measured Conc.Storage Time Conc. Main Peak Conc. Main Peak Conc. Main Peak pH buffer(mg/ml) Temp. (C.) (days) (mg/ml) (area %) (mg/ml) (area %) (mg/ml)(area %) 5.00 citrate 1.0 8 0 0.98, 0.98 100, 100 0.95, 0.95 98.5, 98.50.97, 0.97 98.2, 98.1 — 7 0.98, 0.98 100, 100 0.99, 0.99 98.5, 98.50.97, 0.98 98.5, 98.5 5.05 13 0.97, 0.97 100, 100 0.94, 0.94 98.1, 98.20.96, 0.96 98.2, 98.0 — 19 0.99, 0.99 100, 100 0.95, 0.95 98.1, 98.00.98, 0.98 98.5, 98.6 5.02 27 0.98, 0.99 100, 100 0.99, 0.98 98.0, 98.10.98, 0.98 98.6, 98.6 5.04 41 0.96, 0.96 100, 100 0.94, 0.94 97.5, 97.60.95, 0.96 98.7, 98.6 5.04 55 0.98, 0.98 100, 100 0.94, 0.94 97.0, 97.20.97, 0.98 98.6, 98.8 5.00 citrate 1.0 25 0 0.98, 0.98 100, 100 0.95,0.95 98.5, 98.5 0.97, 0.97 98.2, 98.1 — 7 0.97, 0.97 100, 100 0.97, 0.9797.0, 97.0 0.98, 0.98 98.8, 98.6 5.06 13 0.98, 0.97 100, 100 0.92, 0.9194.6, 94.7 0.97, 0.97 98.8, 98.8 — 19 0.99, 1.00 100, 100 0.90, 0.8992.2, 92.3 0.98, 0.98 98.8, 98.6 5.05 27 0.99, 0.99 100, 100 0.91, 0.9190.3, 90.3 0.99, 0.98 98.8, 98.8 5.06 41 0.97, 0.97 100, 100 0.80, 0.8083.0, 83.0 0.96, 0.96 98.6, 98.7 5.00 55 0.99, 0.98 100, 100 0.76, 0.7677.7, 78.0 0.99, 0.97 99.0, 98.7 Underlined values represent repeatanalyses

TABLE 17 Summary of 1.0 mg/ml, pH 5.0 AM424 Formulation StabilityFollowing Storage at −80° C. and −20° C. Nominal RP - IEC - SEC - AM424Storage Measured RP - Measured IEC - Measured SEC - measured Conc.Storage Time Conc. Main Peak Conc. Main Peak Conc. Main Peak pH buffer(mg/ml) Temp. (C.) (days) (mg/ml) (area %) (mg/ml) (area %) (mg/ml)(area %) 4.96 citrate 1.0 −80 0 1.00, 1.00 100, 100 0.98, 0.99 98.8,98.8 0.98, 0.98 98.1, 98.1 4.97 28 1.00, 0.99 100, 100 0.96, 0.96 98.2,98.1 0.98, 0.98 98.4, 98.4 4.95 56 0.97, 0.97 100, 100 0.95, 0.95 98.4,98.4 0.96, 0.96 98.5, 98.4 4.97 84 0.99, 0.99 100, 100 0.96, 0.96 98.4,98.5 0.97, 0.97 98.5, 98.5 4.96 citrate 1.0 −20 0 1.00, 1.00 100, 1000.98, 0.98 98.8, 98.8 0.98, 0.98 98.1, 98.1 4.96 28 0.99, 1.00 100, 1000.97, 0.97 98.3, 98.2 0.98, 0.98 98.5, 98.4 4.94 56 0.98, 0.97 100, 1000.95, 0.95 98.3, 98.3 0.97, 0.96 98.6, 98.5 4.96 84 0.99, 0.99 100, 1000.96, 0.96 98.4, 98.3 0.97, 0.96 98.5, 98.6

EXAMPLE 2

I. Analytical Methods

-   -   A. Reversed Phase (RP), Ion Exchange (IE) and Size Exclusion        (SEC) Assays were conducted as described in Example 1.        II. Buffer Composition

All LIF samples were prepared by dilution of stock LIF solutioncontaining 3.67 mg/ml LIF in 2 mM phosphate buffer, pH 6.42 to give thedesired final LIF concentration (either 0.4 or 1.0 mg/ml) andcomposition of buffer components. The final composition of each solutioncontained 10 mM citrate buffer, 5% w/v sorbitol and 0.01% w/vPolysorbate 80. Samples differed in the final concentration of phosphatebuffer (present from the original stock LIF solution) depending on thedilution factor. The 0.4 mg/ml LIF solutions contained 0.22 mM residualphosphate while the 1.0 mg/ml LIF solutions contained 0.54 mM residualphosphate. The composition of each buffer was as follows:

A. Citrate Buffer for 0.4 mg/ml LIF Formulations

-   -   Solution A:        -   11.22 mM sodium citrate dihydrate (Merck #1.06448)        -   5.61% w/v sorbitol (Sigma Chemicals #S1876)        -   0.0112% Polysorbate 80 (Sigma Chemicals #P1754)    -   Solution B:        -   11.22 mM citric acid monohydrate (Merck #1.00244)        -   5.61% w/v sorbitol (Sigma Chemicals #S1876)        -   0.0112% Polysorbate 80 (Sigma Chemicals #P1754)

Solutions A and B were mixed to give a final pH of 5.5. Formulationswere prepared by combining 0.109 parts stock LIF solution and 0.891parts buffer to give a final LIF concentration of 0.4 mg/ml, a finalbuffer concentration of 10 mM, a final sorbitol concentration of 5% w/vand a final Polysorbate 80 concentration of 0.01% w/v. The measuredosmolality of the final 0.4 mg/ml LIF formulation was 317 mOsm/kg.

B. Citrate Buffer for 1.0 mg/ml LIF Formulations

-   -   Solution A:        -   13.75 mM sodium citrate (Merck #1.06448)        -   6.88% w/v sorbitol (Sigma Chemicals #S1876)        -   0.0138% w/v Polysorbate 80 (Sigma Chemicals #P1754)    -   Solution B:        -   13.75 mM citric acid (Merck #1.00244)        -   6.88% w/v sorbitol (Sigma Chemicals S1876)        -   0.0138% w/v Polysorbate 80 (Sigma Chemicals P1754)

Solutions A and B were mixed to give a final pH of 5.5. Formulationswere prepared by combining 0.272 parts stock LIF solution and 0.728parts buffer to give a final LIF concentration of 1.0 mg/ml, a finalbuffer concentration of 10 mM, a final sorbitol concentration of 5% w/vand a final Polysorbate 80 concentration of 0.01% w/v. The measuredosmolality of the final 1.0 mg/ml LIF formulation was 322 mOsm/kg.

II. Long Term Stability at 8° C. and 25° C.

A. Preparation of Samples for Storage at 8° C. and 25° C.

LIF formulations were prepared by dilution of stock LIF (3.67 mg/ml in 2mM phosphate buffer, pH 6.42) with citrate buffer containing sorbitoland polysorbate 80 to give a final LIF concentration of 0.4 mg/ml or 1.0mg/ml, a final buffer concentration of 10 mM, a final sorbitolconcentration of 5% w/v and a final polysorbate 80 concentration of0.01% w/v (see Section II). The theoretical pH was 5.5 and the actual pHof each sample was measured and recorded.

Under aseptic conditions in a laminar flow cabinet, the formulationswere sterile filtered using 0.22 μm Millex GV (Millipore) filters. Thefirst 1.15 ml of each filtrate was set aside and the vial markedaccordingly. The remaining volume was filtered into a sterile 50 mlpolypropylene tube. Aliquots of each formulation (1.15 ml/vial) weretransferred using a multiple dispensing Eppendorf pipette with steriletips into heat sterilised 2 ml glass vials and capped with sterileteflon lined rubber caps which were then crimped. Vials were labelledand duplicate vials of each formulation were retained for the initialanalysis. The remaining vials were stored at either 8° C. or 25° C.

B. Sample Analysis

All LIF samples were analysed undiluted along with standards accordingto the methods described in Example 1. At each time point, 2 vials werewithdrawn from the incubators and approximately 200 μl was removed fromeach using a sterile 1 ml syringe and a sterile needle. These aliquotswere placed into polypropylene autosampler vials and sealed with capscontaining self-sealing septa to allow repeat injections from the samevial without evaporation. The original glass sample vials were thenmarked with the time point and placed at −80° C. for repeat analysis (ifrequired) or use in other studies.

Autosampler vials were transferred to the autosampler where they weremaintained at 4° C. throughout the three analytical runs. The samesample and standard autosampler vials were used for each of the threeanalyses with the RP (10 μl injection volume) being conducted first,followed by the IEC (100 μl injection volume) and then the SEC (10 μlinjection volume). The complete RP run took approximately 20 hours, andthe IEC and SEC runs took approximately 15 hours each. It was assumedthat any further degradation over this storage time in the autosamplerwould be minimal (standard solutions at pH 6.42 stored under the sameconditions showed no change over the complete analytical period).

Selected samples were also analysed for particulates using a MalvernInstruments Zetasizer 3000 particle size instrument. Samples werewithdrawn from the storage vials using a syringe and placed in thesample cuvette. Samples were counted for 120 sec using a 200 μm pinhole(to obtain the maximum signal), 90° scattering angle, and scatteringsource at 633 nm using a 10 mW He—Ne ion laser.

IV. Results

Data pertaining to solution pH, LIF concentration in mg/ml (determinedby comparison to LIF standard solutions), and the area % for the mainpeak relative to the total peak area for all LIF related peaks in thechromatogram analysed using the three chromatographic methods are shownin Tables 18 and 19. For each set of samples, there was a slightdecrease in solution pH of approximately 0.1 unit over the 92 daystorage period.

1. Ion Exchange

A single main product (eluting at approximately 9 min) was seen in allsamples stored at 8 and 25°. There was evidence of several minordegradation products in the ion exchange chromatograms, however, due toinadequate resolution between the different products, the exact numberof products could not be determined. Samples prepared at pH 5.0 (initialstudy) and those at pH 5.5 (this study) stored at 8° C. and 25° C. for 8weeks were compared. The chromatograms were normalised with respect tothe retention time for the main peak to take into account slight changesin the chromatography between the two studies. In each case, the productdistribution was similar with a higher proportion of the maindegradation product noted in the pH 5.5 samples relative to the pH 5.0samples.

The IEC results for the samples, wherein the main LIF peak was plottedas a percentage of the total area for all LIF related peaks in thechromatogram as a function of storage time illustrated the dependence ofLIF stability on temperature. The relative stability under each storagecondition was similar for the 0.4 and 1.0 mg/ml formulations. After 92days storage at 8° C., 95–96% of the total peak area was present as themain LIF peak. Following storage at 25° C. for 92 days, this value wasreduced to approximately 56–58%.

The IEC stability data (main peak area expressed as a percentage of thetotal) obtained for samples at pH 5.5 and that from the previous studywith samples prepared at pH 5.0 were compared. At 25° C., a slightincrease in the rate of degradation was evident at pH 5.5.

2. Reversed Phase

RP chromatograms for the samples all displayed essentially the sameelution characteristics. In all cases, the chromatograms showed thepresence of only one main peak eluting at approximately 36 min.

The RP results wherein the measured concentration was plotted as afunction of storage time illustrated the absence of significant changein the measured concentration over the storage period.

3. Size Exclusion

SEC chroratograms for the samples displayed essentially the same elutioncharacteristics. In all cases, the chromatograms showed the presence ofone main peak eluting at approximately 25 min and a minor peak elutingat approximately 21 min.

The SEC results wherein the measured concentration was plotted as afunction of storage time illustrated the absence of significant changein the measured concentration over the storage period. Using the SECmethod, there was no evidence of chain cleavage or crosslinking underthe storage conditions studied.

4. Particle Size Analysis

Samples stored for 102 days at 8 and 25° C. were analysed forparticulates using a laser light scattering instrument. AU of thesamples analysed displayed a count rate of “0–0.5 kCps” whicheffectively means that the samples contained no particulates (i.e. nosignal was measurable).

V. Summary

These studies demonstrated that formulations prepared at pH 5.5 werestable for up to 13 weeks when stored at 8° C. with loss of the parentcompound being approximately 3% as shown by IEC. After storage for 56days at 8° C., the loss of LIF was approximately 2% in comparison toapproximately 1% for pH 5.0 samples stored under the same conditions(data from the initial study). At 25° C., the rate of degradation at pH5.5 was significantly increased with approximately 12% loss occurring in4 weeks. In comparison, pH 5.0 samples showed a decrease in LIFconcentration of approximately 7–9% after 4 weeks at 25° C. As in theinitial study, no loss of LIF was detected by RP or SEC under any of theconditions studied.

TABLE 18 Summary of AM424 Stability for 0.4 mg/ml Formulations at pH 5.5Following Storage at 8° C. and 25° C. RP - IEC - SEC - Nominal StorageMeasured RP - Measured IEC - Measured SEC - Measured LIF Conc. StorageTime Conc. Main Peak Conc. Main Peak Conc. Main Peak pH buffer (mg/ml)Temp. (C.) (days) (mg/ml) (area %) (mg/ml) (area %) (mg/ml) (area %)5.61 citrate 0.4 8 0 0.42, 0.44 100, 100 0.40, 0.40 98.6, 98.6 0.40,0.40 98.7, 98.7 5.56 14 0.38, 0.38 100, 100 0.37, 0.37 97.9, 98.0 0.39,0.39 98.7, 98.7 5.59 29 0.41, 0.41 100, 100 0.39, 0.39 98.4, 98.3 0.41,0.41 98.8, 98.7 5.51 42 0.40, 0.41 100, 100 0.37, 0.38 98.1, 98.7 0.40,0.39 98.6, 98.7 5.47 56 0.39, 0.39 100, 100 0.39, 0.39 97.3, 97.1 0.40,0.40 98.6, 98.7 5.48 77 0.39, 0.40 100, 100 0.38, 0.38 96.2, 96.3 0.39,0.39 98.9, 98.9 5.48 92 0.42, 0.40 100, 100 0.37, 0.37 95.7, 95.8 0.38,0.38 98.6, 98.7 5.61 citrate 0.4 25 0 0.42, 0.44 100, 100 0.40, 0.4098.6, 98.6 0.40, 0.40 98.7, 98.7 5.57 14 0.38, 0.39 100, 100 0.35, 0.3592.8, 92.8 0.39, 0.39 98.8, 98.9 5.59 29 0.41, 0.42 100, 100 0.35, 0.3586.9, 86.8 0.41, 0.41 98.9, 99.0 5.52 42 0.41, 0.41 100, 100 0.31, 0.3281.0, 81.8 0.38, 0.40 98.8, 99.0 5.48 56 0.39, 0.39 100, 100 0.29, 0.2971.6, 71.9 0.41, 0.40 99.2, 99.0 5.48 77 0.41, 0.40 100, 100 0.26, 0.2663.8, 64.0 0.40, 0.39 99.3, 99.1 5.48 92 0.40, 0.42 100, 100 0.23, 0.2357.4, 57.7 0.39, 0.40 98.9, 99.2

TABLE 19 Summary of AM424 Stability for 1.0 mg/ml Formulations at pH 5.5Following Storage at 8° C. and 25° C. RP - IEC - SEC - Nominal StorageMeasured RP - Measured IEC - Measured SEC - Measured LIF Conc. StorageTime Conc. Main Peak Conc. Main Peak Conc Main Peak pH buffer (mg/ml)Temp. (C.) (days) (mg/ml) (area %) (mg/ml) (area %) (mg/ml) (area %)5.61 citrate 1.0 8 0 1.09, 1.08 100, 100 1.00, 1.00 98.6, 98.6 1.01,1.01 98.6, 98.6 5.58 14 0.98, 0.99 100, 100 0.96, 0.96 97.7, 97.7 0.99,0.99 98.6, 98.7 5.61 29 1.01, 1.02 100, 100 0.99, 0.99 97.5, 97.6 1.01,1.01 98.5, 98.5 5.57 42 1.00, 1.01 100, 100 0.97, 0.97 97.3, 97.2 0.99,0.98 98.4, 98.6 5.54 56 1.00, 0.99 100, 100 0.96, 0.96 96.8, 96.6 1.02,1.02 98.4, 98.5 5.52 77 1.03, 1.02 100, 100 0.94, 0.94 96.0, 95.9 0.98,0.99 98.5, 98.5 5.52 92 1.06, 1.04 100, 100 0.95, 0.94 95.3, 95.3 0.98,0.98 98.4, 98.4 5.61 citrate 1.0 25 0 1.09, 1.08 100, 100 1.00, 1.0098.6, 98.6 1.01, 1.01 98.6, 98.6 5.58 14 0.98, 0.98 100, 100 0.90, 0.9091.7, 91.8 0.99, 0.99 98.7, 98.7 5.62 29 1.02, 1.01 100, 100 0.87, 0.8785.6, 85.7 1.02, 1.02 98.7, 98.7 5.59 42 1.02, 1.01 100, 100 0.80, 0.8080.0, 79.8 0.98, 0.98 98.8, 98.8 5.54 56 0.99, 1.02 100, 100 0.71, 0.7168.9, 69.2 1.02, 1.03 98.8, 98.7 5.53 77 1.02, 1.03 100, 100 0.64, 0.6461.0, 61.6 0.98, 0.98 98.9, 98.8 5.52 92 1.04, 1.09 100, 100 0.59, 0.5856.0, 56.1 0.99, 0.99 98.8, 98.7

EXAMPLE 3

I. Sample Preparation

8° C. and 25° C. LIF Samples

LIF formulations were prepared by a dilution of stock LIF (3.67 mg/ml in2 mM phosphate buffer) with citrate buffer containing sorbitol or NaClto give a final LIF concentration of 0.4 mg/ml, a final bufferconcentration of 10 mM, a final sorbitol concentration of 5% w/v or afinal NaCl concentration of 0.9% w/v. The theoretical pH was 5.0 in allcases. Formulations were prepared and filled into vials as describedpreviously.

II. Analytical Methods

Samples and standards were prepared as previously described. Analyseswere conducted by RP and SEC and IEC was conducted using the Polycat Acolumn.

The RP and SEC assays were the same as those described in Example 1. TheIEC assay was conducted using a PolyLC PolyCAT A cation exchange column,pH 6 phosphate buffer and a salt gradient. Detection was at 215 nm.

III. Results

Ion Exchange

IEC data for 0.4 mg/ml formulations are shown in Tables 20 and 21. Theresults expressed as a percentage of the initial concentration remainingafter the storage period indicated that the most stable formulationswere the pH 5.0 citrate buffer containing sorbitol and Tween 80 and thepH 5.0 citrate buffer containing NaCl.

SEC

SEC data for 0.05 and 0.4 mg/ml formulations are plotted with the mainpeak expressed as a % of the total area. There was some variability inthe 0.05 mg/ml samples most likely due to the low concentration. Therewere no real trends for either buffer at 8° C. or 25° C.

Freeze-Thaw Cycling

Freeze-thaw cycling studies for pH 5 citrate buffers containing sorbitolor NaCl were analysed by SEC. After the 5th cycle there was a trendtoward a decrease in the main peak as a % of the total area and a slightincrease in the pre-eluting high molecular weight peak.

TABLE 20 Stability of 0.4 mg/mL LIF formulations following storage at 8°C. measured by IEC. Storage Citrate/Sorbitol/TweenCitrate/Sorbitol/Tween Citrate/Sorbitol pH 5.0 Time pH 5.0 (% of initialconc pH 5.5 (% of initial conc (% of initial conc Citrate/NaCl pH 5.0 (%(Weeks) remaining) remaining) remaining) of initial conc remaining) 0100 100 100 100 2 99.7 99.4 99.9 101.6 4 99.8 99.7 99.7 101.1 6 99.299.2 98.2 98.8 8 98.8 98.5 97.0 98.2

TABLE 21 Stability of 0.4 mg/Ml LIF formulations following storage at25° C. measured by IEC. Storage Citrate/Sorbitol/TweenCitrate/Sorbitol/Tween Citrate/Sorbitol pH 5.0 Time pH 5.0 (% of initialconc pH 5.5 (% of initial conc (% of initial conc Citrate/NaCl pH 5.0 (%(Weeks) remaining) remaining) remaining) of initial conc remaining) 0100 100 100 100 2 96.4 94.1 84.0 91.8 4 93.0 88.0 80.6 86.2 6 88.6 83.072.2 81.5 8 78.9 72.9 68.7 76.0

EXAMPLE 4

Preferred compositions comprise:

-   -   LIF in a concentration of 400 to 1000 mg/ml    -   pH of about 4.0–6.0    -   surfactant    -   isotonicity agent    -   buffer.

Particularly preferred compositions are those wherein the pH range isabout 4.5–5.5.

EXAMPLE 5

A particularly preferred composition comprises:

-   -   LIF in a concentration of 400 to 1000 mg/mil    -   pH of about 5.0    -   5% w/w sorbitol    -   0.01% polysorbate 80    -   citrate or acetate buffer.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

1. A composition comprising leukaemia inhibitory factor (LIF) and astabilizing agent, additives for maintaining pH and isotonicity and oneor more pharmaceutically acceptable carriers wherein the pH of thecomposition is between about 3.5 and 6.5.
 2. A composition according toclaim 1 wherein the stabilizing agent is an agent which increases ormaintains the conformational stability of LIF.
 3. A compositionaccording to claim 2 wherein the stabilizing agent is selected from apolyhydric alcohol, a pharmaceutically acceptable salt, a bufferspecies, a sugar and a pharmaceutically acceptable polymeric compound.4. A composition according to claim 3 wherein the polyhydric alcohol issorbitol.
 5. A composition according to claim 2 wherein the stabilizingagent is an anionic, cationic, amphoteric or non-ionic surfactant.
 6. Acomposition according to claim 5 wherein the surfactant is selected froma fatty alcohol, a glyceryl ester and a fatty acid ester of a fattyalcohol or other alcohol.
 7. A composition according to claim 2 whereinthe stabilizing agent is selected from a polysorbate, a polyoxyethylenederivative and a pharmaceutically acceptablepolyoxyethylene-polyoxpropylene copolymer.
 8. A composition according toclaim 3 wherein the buffer species is selected from a phosphate, citrateand acetate buffer.
 9. A composition according to claim 8 wherein thebuffer species is a citrate or acetate buffer.
 10. A compositioncomprising leukaemia inhibitory factor (LIF), additives for maintainingpH and isotonicity and one or more pharmaceutically acceptable carriersand/or diluents and wherein the composition has a pH of between about3.5 and 6.5.
 11. A composition according to claim 10 wherein theaggregation of LIF is reduced over time.
 12. A composition according toclaim 1 wherein LIF is present in an amount from about 0.1 μg/ml toabout 100 mg/ml.
 13. A composition according to claim 9 wherein LIF ispresent in an amount from about 0.1 μg/ml to about 100 mg/ml.
 14. Acomposition according to claim 1 wherein the pH of the composition isbetween about 4.5 and 6.5.
 15. A composition according to claim 14wherein the pH of the composition is between about 4.5 and 6.0.
 16. Acomposition according to claim 2 wherein the stabilizing agent is asurfactant.
 17. A composition according to claim 5 wherein thestabilizing agent is polysorbate 20 and/or polysorbate
 80. 18. Acomposition according to claim 10 wherein the composition has a pH ofbetween about 4.5 and 6.5.
 19. A composition according to claim 18wherein the composition has a pH of between about 4.5 and 6.0.
 20. Acomposition according to any one of claims 1, 14 or 15 wherein thestabilizing agent facilitates reduced aggregation of LIF.
 21. Acomposition according to any one of claims 1, 14 or 15 wherein thestabilizing agent facilitates a reduction in the deamidation of LIF. 22.A composition according to any one of claims 10, 18 or 19 wherein thedeamidation of LIF is reduced over time.
 23. A composition according toany one of claims 10, 18 or 19 where the pH is maintained by thepresence of a buffer species selected from a phosphate, citrate andacetate buffer.
 24. A composition according to claim 23 wherein thebuffer species is a citrate or acetate buffer.
 25. A method forpreparing a composition comprising leukaemia inhibitory factor (LIF)wherein said composition exhibits reduced deamidation and/or aggregationof LIF over times said method comprising admixing LIF with a stabilizingagent and additives for maintaining pH and isotonicity.
 26. A methodaccording to claim 25 wherein the stabilizing agent is an agent whichincreases or maintains the conformational stability of LIF or is asurfactant.
 27. A method according to claim 26 wherein the stabilizingagent is selected from a polyhydric alcohol, a pharmaceuticallyacceptable salt, a buffer species, a sugar and a pharmaceuticallyacceptable polymeric compound.
 28. A method according to claim 27wherein the polyhydric alcohol is sorbitol.
 29. A method according toclaim 26 wherein the surfactant is an anionic, cationic, amphoteric ornon-ionic surfactant.
 30. A method according to claim 29 wherein thesurfactant is selected from a fatty alcohol, glyceryl ester and a fattyacid ester of a fatty alcohol or other alcohol.
 31. A method accordingto claim 25 wherein the additives for maintaining pH and isotonicty areselected from a phosphate, citrate and acetate buffer.
 32. A methodaccording to claim 31 wherein the additives for maintaining pH andisotonicity are citrate or acetate buffer.
 33. A method according toclaim 25 further comprising adjusting the pH to between about 3.5 andabout 6.5.
 34. A method according to claim 25 further comprisingadmixing at least one of a pharmaceutically acceptable carrier ordiluent.
 35. A method according to claim 27 wherein the stabilizingagent is selected from a polysorbate, a polyoxyethylene derivative and apharmaceutically acceptable polyoxyethylene-polyoxypropylene copolymer.36. A method according to claim 35 wherein the polysorbate ispolysorbate 20 and/or polysorbate
 80. 37. A method according to claim 33further comprising adjusting the pH to between about 4.5 and about 6.5.38. A method according to claim 33 further comprising adjusting the pHto between about 4.5 and 6.0.
 39. A method of preparing a compositioncomprising leukemia inhibitory factor (LIF) wherein said compositionexhibits improved chemical or physical stability of LIF, said methodcomprising admixing LIF or its derivative or homologue with astabilizing agent.
 40. A method according to claim 39 wherein thestabilizing agent is selected from a polyhydric alcohol, apharmaceutically acceptable salt, a buffer species, a sugar and apharmaceutically acceptable polymeric compound.
 41. A method accordingto claim 40 wherein the polyhydric alcohol is sorbitol.
 42. A methodaccording to claim 40 wherein the stabilizing agent is an anionic,cationic, amphoteric or non-ionic surfactant.
 43. A method according toclaim 42 wherein the surfactant is selected from a fatty alcohol,glyceryl ester and a fatty acid ester of a fatty alcohol or otheralcohol.
 44. A method according to claim 39 where the stabilizing agentis selected from a polysorbate, a polyoxyethylene derivative or apharmaceutically acceptable polyoxyethylene-polyoxypropylene copolymer.45. A method according to claim 40 wherein the buffer species isselected from a phosphate, citrate and acetate buffer.
 46. A methodaccording to claim 45 wherein the buffer species is a citrate or acetatebuffer.
 47. A method according to claim 39 where the pH of thecomposition is between about 3.5 to about 6.5.
 48. A method according toclaim 47 wherein the pH is between about 4.5 and about 5.5.
 49. A methodaccording to claim 48 wherein the pH is between about 4.5 and about 6.0.